KR101668609B1 - composition containing leelamine for inducing cytochrome P450 2B or selectively inducing cytochrome P450 2B - Google Patents

Abstract

The present invention relates to a composition for inducing CYP2B enzyme containing lelamine as an active ingredient. The present invention relates to a novel specific drug metabolizing enzyme composition for inducing the activity of CYP 2B enzyme and exhibiting selective inducing activity against CYP 2B which is not involved in other CYP activity, Drug metabolism studies for prediction of action and co-administration.

Description

[0001] The present invention relates to a cytochrome P450 2B enzyme-inducing and selective inducing composition containing as an active ingredient lelamine,

The present invention relates to a composition for inducing and selectively inducing cytochrome P450 2B enzyme containing lelamine as an active ingredient.

Drug metabolism in the liver plays a pivotal role in the excretion of exogenous substances that are absorbed, and this response is mediated by a drug metabolizing enzyme system consisting of normal metabolism and abnormal metabolizing enzymes. Expression and activity of drug metabolizing enzymes are induced or repressed by exposure of exogenous substances, which is a major cause of drug interaction. In addition, pathological physiological conditions such as diabetes, obesity, malnutrition, drinking, and inflammation have been reported to regulate the expression and activity of drug metabolizing enzymes in the liver.

Drug interactions are a typical side effect of drugs that are caused by the change of drug efficacy and pharmacokinetics by the combined use of drugs that regulate the activity of CYP. Recently, the number of patients who are taking long-term multidrug therapy together with the development of various drugs is increasing. Therefore, it is important to predict drug interactions to minimize side effects and to maximize pharmacological action. Cytochrome p450 (CYP450) is involved in the oxidative metabolism of endogenous substances such as steroid hormones, fatty acids, prostaglandins and exogenous substances (especially drugs) It is a group of enzymes containing heme that convert to a hydrophilic material for easy excretion by urine or bile. It is found in all plants and animals. It is mainly present in the membranes of the endoplasmic reticulum of the hepatocytes and is present in small amounts in the small intestine, kidney, lung, and brain.

CYP450 reduced the microsomes to NADH and dithionide (hydrosulfide) in 1958, and after passing through carbon monoxide, discovered that there was a dye showing maximum absorbance at 450 nm (Klingenberg M., Pigments Arch. Biochem Biophys., 75 (2), pp. 376-86, 1958), which revealed that this was a cytochrome-type heme protein and named it CYP450 (Omura T, Sato R. , A cytochrome in liver microsomes, J Biol Chem., 237, pp. 1375-6, 1962). The nomenclature of CYP450, which is widely used at present, is classified into three stages based on the similarity of amino acid sequence order. CYP450 is classified into three stages by adding the Arabic numerals, the upper case letters and the Arabic numerals to the CYP prefixes and the subfamily, (Isoenzyme). For example, CYP3A4 refers to family 3, subfamily A, and isoenzyme 4. In this case, the amino acid sequence is about 40% or more similar to the amino acid sequence, and the subunit is a group having the same amino acid sequence of 55% or more.

CYP450 genetically shows phenotype and genotype as a genetic polymorphism in the population. To date, more than 30 CYP450 isozymes have been found in humans, and it is known that CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C19, and CYP2E1 are mainly involved in drug metabolism. These are important factors influencing the clinical, therapeutic and side effects of substrate drugs metabolized by these kinases in individual patients. CYP450 inhibition means that one drug (inhibitor) decreases the activity of the enzyme and consequently increases the blood concentration of the other drug, the substrate of the enzyme. In addition, induction of CYP450 occurs when hepatic blood flow increases or CYP450 production increases due to a specific drug or environmental pollutant, and CYP450 increases the capacity to metabolize the drug, It means to decrease the blood concentration.

On the other hand, specific CYPs induce changes in metabolic efficiency of other drugs and the resulting side effects account for 30% of drug interactions due to metabolic enzymes. CYP 2B is a typical CYP isoform that is strongly induced by external drugs and is involved in drug interactions. Thus, the induction of CYP 2B is an important indicator in evaluating the inducibility of candidate substances during the development of new drugs, and the induction ability of candidate substances is compared with that of phenobarbital or rifampin, . Two substances used as control substances have been reported to have similar inducibility to CYP 2B as well as other CYPs. Such nonselective inducing effect has been recognized as a problem to be solved in the evaluation of candidate substances, but there is no research on this.

The present inventors have studied to develop a composition having an excellent effect of selectively inducing CYP 2B, and found that lelamine exhibits a more potent CYP 2B-inducing effect than the conventional drug metabolizing enzyme inducing agent and does not participate in other CYP activity, Selective inducing ability, and completed the present invention.

It is an object of the present invention to provide a composition for inducing and selectively inducing cytochrome P450 2B enzyme containing lelamine as an active ingredient.

It is also an object of the present invention to provide a positive control for evaluating cytochrome P450 2B enzyme-inducing ability including lelamine.

It is also an object of the present invention to provide a kit for evaluating the cytochrome P450 2B enzyme-inducing ability including the positive control.

It is also an object of the present invention to provide a method for evaluating cytochrome P450 2B enzyme-inducing ability of a candidate drug using lelamine as a positive control.

In order to accomplish the above object, the present invention provides a composition for inducing and selectively inducing cytochrome P450 2B enzyme comprising lelamine as an active ingredient.

In addition, the present invention provides a positive control for evaluating the cytochrome P450 2B enzyme-inducing ability including lelamine.

In addition, the present invention provides a kit for evaluating cytochrome P450 2B enzyme-inducing ability including the positive control group.

The present invention also provides a method for evaluating cytochrome P450 2B enzyme-inducing ability of a candidate drug using lelamine as a positive control.

The present invention relates to a novel specific drug metabolizing enzyme composition for inducing the activity of CYP 2B enzyme and exhibiting selective inducing activity against CYP 2B which is not involved in other CYP activity, Drug metabolism studies for prediction of action and co-administration.

Figure 1 shows the effect of lelamine on the activity of BROD and PROD, the CYP 2B dependent enzymes.
Figure 2 shows the effect of lelamine on CYP2B protein levels.
Figure 3 shows the effect of lelamine on mRNA levels of CYP2B.
Figure 4 shows the effect of lelamine on the degradation of CYP2B.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for inducing cytochrome P450 2B enzyme comprising lelamine as an active ingredient represented by the following formula (1).

[Chemical Formula 1]

The effective ingredient of the present invention, lelamine, can be synthesized by a conventional organic synthesis method or a commercially available reagent can be purchased and used.

The cytochrome P450 2B (hereinafter referred to as CYP 2B) of the present invention is a subtype of cytochrome P450 (CYP) as a drug metabolizing enzyme.

The lelamine of the present invention strongly induces the activity of the CYP 2B enzyme and has an inducing effect of CYP 2B enzyme which is superior to that of the conventional drug metabolizing enzyme inducing agent phenobarbital or rifampin.

In addition, the composition for inducing CYP2B enzyme of the present invention may be a selective inducer of CYP2B enzyme which is not involved in other CYP activity.

The conventional drug metabolizing enzyme-inducing agent was involved not only in CYP 2B but also in other CYPs, and showed a non-selective inducing effect. However, the composition of the present invention has a selective inducing effect of CYP 2B enzyme and is useful as a composition for inducing a new drug metabolizing enzyme And may be useful for drug metabolism studies and co-administration for predicting drug interactions.

The composition of the present invention may further contain at least one known active ingredient having a CYP 2B-inducing effect together with lelamine.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the lelamine of the present invention may be administered in an amount of 0.01 mg / kg to 100 mg / kg per day, and may be administered once or several times a day. The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The compositions of the present invention can be used alone or in combination with methods for the induction of drug metabolizing enzymes, or using methods of surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The present invention also provides a positive control for evaluating the cytochrome P450 2B (CYP 2B) enzyme-inducing ability, comprising lelamine or a pharmaceutically acceptable salt thereof represented by the following formula (1). The lelamine of the present invention can be used not only to induce CYP enzymes but also to evaluate the ability of candidate substances to induce CYP enzymes as positive controls.

[Chemical Formula 1]

The positive control of the present invention may be a positive control indicating selective inducibility of CYP2B enzyme not involved in other CYP activities.

In addition, the present invention provides a kit for evaluating cytochrome P450 2B enzyme-inducing ability including the positive control group.

These kits may include not only the positive control of the present invention but also tools, reagents and the like commonly used in the art used for analyzing drug metabolizing enzyme activity. Such tools / reagents may include, but are not limited to, suitable carriers, labeling agents capable of generating detectable signals, solubilizers, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers such as, for example, physiologically acceptable buffers (PBS), insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, Crosslinked dextran, polysaccharides, polymers such as magnetic microparticles plated with metal on latex, other species, glass, metal, agarose, and combinations thereof.

The present invention also provides a method for evaluating cytochrome P450 2B enzyme-inducing activity of a candidate drug using lelamine or its pharmaceutically acceptable salt represented by the following formula (1) as a positive control.

[Chemical Formula 1]

The method comprises:

(a) orally administering each of lelamine or a candidate substance to an experimental animal;

(b) measuring cytochrome P450 2B enzyme activity in the laboratory animal to which the relramin is administered;

(c) measuring cytochrome P450 2B enzyme activity in an experimental animal to which the candidate substance is administered; And

(d) judging the candidate substance as a cytochrome P450 2B enzyme inducer when the activity of step (c) is higher than the activity of step (b).

The term "candidate substance " used in the present invention means a substance to be tested for the activity of inducing CYP enzyme, and any molecule such as an extract, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, . Such candidate materials also include synthetic materials as well as natural materials.

In step (a), oral administration of the test compound to the experimental animal can be carried out by any method known in the art without limitation. Such experimental animals include, but are not limited to, animals known in the art such as mice, rats, rabbits, guinea pigs, dogs, cats, monkeys, and the like.

In step (b), the CYP enzyme activity of lelamine-treated experimental animals was measured in order to compare the degree of activation of CYP enzymes in experimental animals treated with lelamine and the degree of activation of CYP enzymes in experimental animals treated with candidate substances to be. Lelamine-treated experimental animals are used as positive control.

Measurement of CYP enzyme activity can be performed by any method known in the art for measuring CYP enzyme activity without limitation, and can be measured, for example, by Western blotting.

Step (d) is a step of judging the candidate substance as a CYP enzyme activity inducing substance when the CYP enzyme activity of step (c) is similar or increased compared to the activity level of step (b).

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, experimental examples and preparation examples.

Example 1. Experimental Materials and Experimental Animals

1-1. Preparation of experimental material

For the oral administration to mice, the refined leelamine hydrochloride was prepared. Phenobarbital Tab. Was purchased from Hana Pharmaceutical Co., Ltd. (Hwasung, Korea). Bovine serum albumin, benzyloxyresorufin, pentoxyresorufin, glucose-6-phosphate, NADPH and glucose-6-phosphate dehydrogenase, 6-phosphate dehydrogenase) was purchased from Sigma Chemical Company (St. Louis, MO). LC grade water and acetonitrile (CAN) were purchased from Merck KGaA (Darmstadt, Germany). Antibodies against human CYP2B6 (OriGene Technologies, Inc., Rockville, USA) were used for detection of heterologous homologous mouse proteins. HRP (horseradish peroxidase) -binding secondary antibody and beta-actin for primary antibody IgG were purchased from Cell Signaling Technology, Inc. (Danvers, USA). All chemicals were of analytical grade.

1-2. Experimental animals and treatment methods

A non-diseased male ICR mouse was purchased from Orient Bio Inc. (Seongnam, Korea) which maintained a strict 12 hour / cancer cycle at 23 ± 3 ° C, 40-60% relative humidity, and 150-300 Lux intensity A 7-week-old mouse was used. Prepared urelamine 5, 10, or 20 mg / kg in physiological saline was orally administered to mice for 1 or 3 consecutive days. Twenty-four hours after the last administration, all mice were autopsied. The liver was removed and homogenized with cold 0.1 M potassium phosphate buffer (pH 7.4). The liver S9 fraction isolated by fractional centrifugation at 9,000 g was stored at -80 [deg.] C until used. S9 protein content was determined using bovine serum albumin as a standard for protein quantification. To compare the metabolic activation intensity of lelamine, male ICR mice were pretreated in PBS (phosphate buffered saline) for 3 days. All animals were treated according to the Guidelines for the Management and Use of Laboratory Animals issued by Kyungpook National University.

Experimental Example 1. Measurement of body weight and liver change of lelamine-treated mice

In order to confirm the effect of lelamine on the expression of CYP, the body weight and liver weight changes of orally administered mice were confirmed, and liver function (ALT) test was carried out, and the results are shown in Table 1 below.

Day Lelamine (mg / kg) Weight (g) Liver weight (g) Weight Loss Weight (%) ALT (karmen unit / ml) One


VH 28.2 ± 0.6 2.3 ± 0.1 8.2 ± 0.4 25.9 ± 1.4 5 26.4 ± 1.4 2.0 ± 0.1 7.8 ± 0.2 25.6 ± 0.5 10 27.6 ± 1.0 2.4 ± 0.1 8.8 ± 0.5 26.9 ± 0.3 20 26.1 ± 0.5 2.4 ± 0.1 9.1 ± 0.3 26.4 ± 0.4 3


VH 33.8 ± 0.4 2.0 ± 0.1 6.0 ± 0.1 22.5 ± 8.7 5 34.5 ± 0.4 2.2 ± 0.1 6.2 ± 0.2 23.3 ± 3.7 10 34.1 ± 0.3 2.3 ± 0.1 6.7 ± 0.2 23.4 ± 2.0 20 32.4 ± 0.5 2.3 ± 0.1 7.2 ± 0.3 23.6 ± 2.0

As shown in Table 1, no significant change in body weight was observed, but liver weight and body weight-to-liver weight slightly increased dose-dependently at both 1 and 3 days of administration. In addition, it was judged that there was no hepatotoxicity by lelamine in the dose used in the experiment.

Experimental Example 2. Measurement of CYP activity using a cocktail probe assay

To measure CYPs activity, a cocktail probe assay was used. The probe substrate and CYP subtypes used are shown in Table 2 below.

temperament The CYP450 subtype (isoform) Phenacetin O-deethylation < RTI ID = 0.0 > CYP1A2 Bupropion hydroxylation < RTI ID = 0.0 > CYP2B Omeprazole 5-hydroxylation (Omeprazole 5-hydroxylation) CYP2C Dextromethorphan O-demethylation (Dextromethorphan O-demethylation) CYP2D Chlorzoxazone 6-hydroxylation (Chloroxazone 6-hydroxylation) CYP2E Midazolam 1-hydroxylation < RTI ID = 0.0 > CYP3A

Specifically, a 0.1 M potassium phosphate buffer solution (pH 7.4), a cocktail probe substrate (see Table 1 below), 0.1 M glucose-6-phosphate, 10 mg / ml? -NADP ⁺ and 1.0 U / ml glucose- A culture mixture containing NGS (NADPH-generation system) containing dehydrogenase was prepared.

The culture mixture was preincubated at 37 ° C for 5 minutes, NGS was added, and the reaction was allowed to proceed for 60 minutes. The reaction was terminated by the addition of 200 μl acetonitrile containing 0.1% formic acid and 5 μl of internal standard solution (10 μM reserpine) to methanol. After mixing and centrifugation at 13,000 × g for 5 minutes, 10 μl aliquots were injected into a C ₁₈ column to perform LC-MS / MS analysis. The results are shown in Table 3.

Day Lelamine
(mg / kg) Remaining Active (%) CYP1A2 CYP2B CYP2C CYP2D CYP2E1 CYP3A One VH 100 ± 9.3 100 ± 12.1 100 ± 6.1 100 ± 5.6 100 ± 6.6 100 ± 4.7 5 119.0 + - 14.7 300.5 ± 17.8 ** 112.6 ± 5.3 ** 78.7 ± 7.3 82.6 ± 9.5 87.5 ± 8.1 10 174.5 ± 25.6 * 380.8 ± 24.5 ** 143.6 ± 11.3 ** 74.0 + - 5.4 111.6 ± 15.8 96.3 ± 3.9 20 111.9 ± 17.8 368.4 ± 59.9 ** 138.4 ± 37.2 78.2 ± 11.2 78.6 ± 8.0 110.7 + - 8.7 3 VH  100 ± 15.7 100 ± 32.8  100 ± 17.1 100 ± 14.9 100 ± 17.0 100 ± 19.3 5 117.7 ± 12.1 1072.2 ± 112.7 ** 133.5 ± 10.4 69.2 ± 5.2 ** 85.0 7.9 92.9 ± 13.0 10 131.1 ± 9.4 1166.3 ± 88.2 ** 139.4 ± 9.4 ** 61.2 ± 4.2 ** 85.9 ± 6.9 82.6 ± 13.5 20 96.4 ± 14.4 825.3 + - 116.9 ** 94.9 ± 13.0  38.0 ± 4.8 ** 71.7 ± 7.9 ** 59.0 ± 13.9 Phenobarbital
(80 mg / kg) 122.9 ± 23.2 269.7 ± 46.5 ** 89.4 ± 14.5 62.9 ± 10.4 ** 88.5 ± 14.7 71.1 + - 12.3 **

As shown in Table 3, CYP2B-catalyzed bupropion 1'-hydroxylation was significantly increased to 269% when treated with lelamine, compared with the PB treated positive control, whereas CYP 1A2, 2C, 2D, 2E1 And other CYPs including 3A were not effective in inducing or inhibiting CYP2B, and it was confirmed that there was an excellent selective activity inducing effect only for CYP2B.

Experimental Example 3: PROD and BROD analysis.

(TECAN infinite 200 pro, Tecan, Switzerland) to determine the effect of lelamine on the activity of the CYP450 2B dependent enzyme BROD (7-benzyloxyresorufin O-dealkylase) and PROD (7-pentoxyresorufin O- ) Was used to measure the activity according to the method of Lubet. The reaction solution (200 ml) contained 2 mg / ml bovine serum albumin (BSA), 5 mM glucose-6-phosphate, 1 U glucose-6-phosphate dehydrogenase, 5 μM NADPH and 2.0 μM 7-benzyl And 0.1 M potassium phosphate buffer (pH 7.4) containing oxyresorufin and pentoxy resorufin. The resorufin was quantified by measuring the excitation maximum at 550 nm and the emission maximum at 585 nm using a fluorescence spectrometer, which is shown in FIG.

As shown in Fig. 1, it was confirmed that the activity of BROD and PROD was induced dose-dependently on lelamine.

Experimental Example 4. Analysis of protein and mRNA levels of CYP 2B

10 μg of the diluted S9 fraction protein was electrophoresed on 12% separating gel using the microsomal fraction (S9 fraction) obtained by centrifugation after liver disruption and cirrhotic disruption of the mouse after administration of the test substance Afterwards, the protein was electrophoretically transferred to a PVDF membrane (PVDF Western blotting membranes, Roche, Germany). The membrane was blocked with 5% BSA in TBST (mixture of Tris-Buffered Saline and Tween 20) (2 hr, RT). The membrane was then incubated overnight at 4 ° C with the primary antibody (CYP2B6, mAb, Origene or β-actin, mAb, Cell signaling) with 5% BSA in TBST. The membrane was washed 5 times with TBST. Horseradish peroxidase-conjugated secondary antibody (Anti-Mouse IgG or Anti-rabbit IgG, HRP-linked) with 3% BSA in TBST (1: 1,000 dilution) Antibody, Cell signaling). The membranes were washed five times with TBST and treated with ECL reagent (Amersham ECL Western blotting analysis system, GE healthcare, UK) and imaged using an ImageQuant LAS 4000 mini biomolecular imager (GE healthcare, UK) Respectively.

As shown in Fig. 2, CYP2B1 protein levels were significantly increased during 1 day and 3 days in relamine dose-dependently. The increase in CYP2B1 protein by lelamine treatment was compared with the increase in CYP2B1 protein by PB treatment, which is a control group, to confirm the inducibility of lelamine.

The results of the electrophoresis analysis of the PCR product were confirmed and are shown in FIG. As shown in FIG. 3, when the treatment with lelamine was conducted for 3 days, the intensity of the CYP2B1 band was more inducible than that of the case treated for 1 day. However, the electrophoretic analysis of the PCR products revealed that There was no significant change in CAR and CYP2B mRNA levels. This means that lelamine does not exhibit CYP2B1 inducing activity by transcription.

Experimental Example 5. Protein degradation assay [

In order to measure the degradation sensitivity of CYP2B in the treatment with lelamine, it was confirmed by proteolytic analysis. The degradation reaction mixture (50 μl) contained 45 μl mouse-S9 fraction and 5 μl of 10 × ATP reaction mixture (0.6 mM ATP, 1.2 mM MgCl ₂ · H ₂ O and ₁ × mixture containing 50 mM Tris-HCl, pH 7.4) . The reaction mixture was incubated with shaking for 18 hours at 37 < 0 > C. After incubation, the reaction mixture protein was quantitated by Bradford assay. Then, 5 μl of 1 × SDS sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 0.5 M DTT, 0.02% bromophenol blue) was added to 10 μg of the reaction mixture, I boiled and prepared an immunization blot. CYP2B of the S9 fraction of each sample was confirmed by immunoblotting with a specific antibody against human CYP2B6, and the results are shown in FIG.

As shown in FIG. 4, the intensity of the CYP2B1 band was increased in a dose dependent manner. Particularly, when 20 mg / kg of lelamine was administered for 3 days, the intensity of the CYP2B band was considerably higher Respectively. Indicating that lelamine inhibits CYP2B degradation in a dose-dependent manner.

Hereinafter, the production example of the present invention will be described.

Formulation Example 1. Preparation of pharmaceutical preparations

1. Manufacturing of powder

Leleamine 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Lelamine 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

Lelamine 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

Lelamine 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

Leleamine 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Claims (7)

A composition for evaluating cytochrome P450 2B (CYP 2B) enzyme-inducing ability, comprising leelamine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

The composition according to claim 1, wherein the composition does not participate in the activity of CYP other than cytochrome P450 2B. A kit for evaluating cytochrome P450 2B (CYP 2B) enzyme inducibility, comprising leleamine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as a positive control.
[Chemical Formula 1]

4. The kit according to claim 3, wherein the lelamine or a pharmaceutically acceptable salt thereof exhibits selective inducing ability against P450 2B enzyme. delete Treating a biological sample separated from an experimental animal or an experimental animal excluding human by using lelamine or a pharmaceutically acceptable salt thereof represented by the following formula 1 as a positive control having cytochrome P450 2B enzyme-inducing activity; And evaluating the cytochrome P450 2B enzyme-inducing ability of the candidate drug and screening the candidate drug.
[Chemical Formula 1]

7. The method of claim 6,
(a) treating a biological sample separated from an experimental animal or an experimental animal enalamine and a candidate substance, excluding a human, respectively;
(b) measuring the cytochrome P450 2B enzyme activity in the sample treated with the relamine or the animal;
(c) measuring cytochrome P450 2B enzyme activity in an experimental animal to which the candidate substance is administered; And
(d) determining the candidate substance as a cytochrome P450 2B enzyme inducing substance when the activity of step (c) is higher than the activity of step (b), and determining the candidate substance as cytochrome P450 2B enzyme Assessing inducibility and screening for candidate drugs.

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